mdi Micro Gel Extraction Kit User Guide ADVANCED MICRODEVICES PVT. LTD. Membrane Technologies 21, Industrial Area, Ambala Cantt (INDIA)

Transcription

1 Micro Gel Extraction Kit User Guide ADVANCED MICRODEVICES PVT. LTD. 21, Industrial Area, Ambala Cantt (INDIA) ADVANCED Tel: , MICRODEVICES , PVT. LTD. 21, Fax: Industrial , Area, Ambala Cantt (INDIA) Tel: , mdi@vsnl.com , , Fax: , Website: info@mdimembrane.com, mdi Membrane Technologies

2 Table of Contents Page 1. Introduction 3 2. Applications 3 3. Storage Conditions 3 4. Quality Assurance 4 5. Safety Information 4 6. Lot Release Criteria 4 7. Technical Support 5 8. Kit contents 5 9. Specifications Principle Important Points to be Considered mdi Micro Gel Extraction Procedure Protocol Trouble Shooting Guide Product Use Limitations Product Warranty and Satisfaction Guarantee Ordering Information 16 Page 2 of 16

3 1. Introduction mdi Micro Gel Extraction Kit is a quick, convenient and economical tool to obtain highly concentrated DNA fragments (70bp-4kb) after gel analysis from standard or low melt Agarose gel. The buffer system provided in the kit facilitates efficient binding of DNA fragments from solublised gel slice onto the spin column. Washing is done with help of wash buffers in order to remove agarose gel, salts, and other impurities from DNA samples. Purified DNA is eluted in small volumes (10µl) of low salt buffer or water for variety of downstream applications. 2. Applications 1. Automated Fluorescent Sequencing 2. Radioactive Sequencing 3. Restriction Digestion 4. Ligation 5. Transformation 6. Cloning 7. PCR 3. Storage Conditions As soon as you receive the kit, open it and store Extraction spin columns at 2-8 C. mdi Micro Gel The remaining kit components can be stored at room temperature. The kit is stable for 1 year at given storage conditions. If precipitates appear in buffers re-dissolve at 37 C for few minutes. Page 3 of 16

4 4. Quality Assurance The mdi Micro Gel Extraction kit is designed for various pre-determined specifications and user requirements such as yield, purity, ruggedness, shelf life and functional convenience. These are produced through a well defined quality management system certified by Underwriters Laboratories, USA for ISO 9001: 2008 which ensures intra lot as well as lot to lot consistency. 5. Safety Information The buffers and the reagents may contain irritants, so wear lab coat, disposable gloves and protective goggles while working with themicro Gel Extraction kit. 6. Lot Release Criteria Each lot of Micro Gel Extraction kit is tested against predetermined specifications to ensure consistent product quality. Page 4 of 16

5 7. Technical Support At mdi, customers are our priority. We will share our experiences to assist you to overcome problems in general product usage as well as customize products for special applications. We will * Stimulate problems, and suggest alternative methods to solve them. * Make changes/ improvements in our existing products/protocols. * Develop special new products and system especially to satisfy your needs. We welcome your feedback to improve our products. 8. Kit Contents Spin Columns Contents Collection Tubes Buffer MMG Buffer GW Buffer GE Hand Book ml Quantity ml ml Storage Temperature 2-8 C Certificate of Quality RT RT RT RT Page 5 of 16

6 9. Specifications Capacity of column reservoir 800µl Binding capacity of membrane (ds DNA) 5µg Recovery of DNA (70bp-4kb) 80% Maximum weight of gel slice 400mg Minimum elution volume for conc. DNA 10µl Total eluate volume 9µl Page 6 of 16

7 10. Principle The mdi Micro Gel Extraction kit allows isolation of high ultra pure DNA fragments from solublised agarose gel which involves: 1. Capturing of DNA from solublised gel slice on spin column 2. Washing 3. Elution 1. Capturing of DNA from solublised gel slice on spin column In order to facilitate adsorption of DNA fragment on to the spin column, suitable ph and salt concentration are required which is achieved by addition of binding buffer MMG. 2. Washing Subsequent to DNA binding from solubilised gel slice on spin column unwanted components like dyes, salts and agarose are washed away. Washing is done by buffer GW. 3. Elution Salt concentration and ph of elution buffer is important for elution efficiency, elution occurs at basic conditions and low salt concentration in small volumes. Elution is done with buffer GE. Page 7 of 16

8 11. Important Points to be Considered Optimization of Binding Buffers The binding buffers should posses appropriate salt concentration and ph to facilitate the efficient binding of single or double stranded DNA (70bp - 4kb) from solublized gel slice on spin column. Centrifugation * All centrifugation steps should be carried out at room temperature at > 10,000 rpm in a conventional table top microcentrifuge. In case of choking of spin column, increase centrifugation time and centrifugation speed to 12000rpm. Check the dissolution of the gel before addition to the column to avoid choking of the spin column Washing * To remove residual wash buffer spin the column for 1-2 minutes at >10,000 rpm. * Flow through should be properly discarded before additional centrifugation step otherwise it will not be removed & may inhibit enzymatic reactions. Elution * Elution buffer should be dispensed on to the center of column for maximum elution efficiency. If eluate volume is lower, then centrifuge for one minute extra at > 10,000 rpm. Yield DNA yield depends on following factors: 1. Volume of elution buffer 2. Amount of starting material Page 8 of 16

9 12. mdi Micro Gel Extraction Procedure Solubilized Gel Slice Bind Wash Dry Elute Ultra Pure DNA Fragment Page 9 of 16

10 13. Protocol 1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel or blade. (Minimize the size of the gel slice by removing extra agarose). 2. Weigh the gel slice in a colorless tube. Add 3 volumes of buffer MMG to 1 volume of gel. (100mg ~ 100µl) For example, add 300µl of buffer MMG to each 100mg of gel. 3. Incubate at 50 C in a water bath for 10 minutes (or untill the gel slice has completely dissolved). To help dissolve the gel, mix by vortexing the tube every 2-3 minutes, during incubation. 4. After the gel slice has dissolved completely, add 1 gel volume of Isopropanol to the sample and mix by inverting the tube. For example, if the agarose gel weight is 100 mg, add 100µl isopropanol. 5. Place the spin column in the collection tube in a suitable rack. To bind DNA, load the sample to the spin column and centrifuge for 1 minute at >10,000 rpm. The maximum volume of the column reservoir is 800µl. For sample volumes of more than 800µl, simply load, balance micro centrifuge and spin again. 6. Discard the flowthrough, and using the same collection tube, wash the spin column with 500µl of buffer MMG by centrifuging for 1 minute at >10,000 rpm. 7. Discard the flowthrough, and using the same collection tube, wash the spin column with 750µl of buffer GW by centrifuging for 1 minute at >10,000 rpm. 8. Discard the flowthrough, place the spin column in the same collection tube and centrifuge for 1-2 minutes at >10,000 rpm. Page 10 of 16

11 Important: This step is necessary to remove residual wash buffer. Residual wash buffer will not be completely removed unless the flow through is discarded before this additional centrifugation. Residual wash buffer may inhibit subsequent enzymatic reactions. 9. Place the spin column in a fresh 1.5ml microfuge tube (Not provided). Elute the DNA by dispensing 10µl of elution buffer GE directly on to the center of the column membrane, allow it to stand for 1 minute and centrifuge for 1 minute at >10,000 rpm. Note: The elution buffer should be dispensed on to the center of the column membrane for maximum recovery. The average eluate volume is 9µl from 10µl elution buffer. If the eluate volume is lower, then centrifuge for one minute extra at >10,000 rpm. Page 11 of 16

12 14. Trouble Shooting Guide A. Little or no Yield of DNA 1. Gel slice not solubilized Mix thoroughly with buffer MMG by completely vortexing the tube every 2-3minutes during 50 C incubation. Increase incubation time to 15 minutes. Preferably incubation should be done in a water bath instead of an oven. 2. Gel slice >400 mg Use multiple columns for gel slices > 400 mg. 3. ph of electrophoresis buffer too high Check the ph of electrophoresis buffer before use. In case it is > 8, prepare fresh buffer. The electrophoresis buffer, if incorrectly prepared or used repeatedly, results in sample ph that exceeds the buffering capacity of buffer MMG and leads to inefficient DNA binding. 4. Improper dispensing of elution buffer Elution buffer must be dispensed on to the center of the column membrane for maximum elution efficiency. 5. Insufficient incubation of elution buffer in the spin column Increase incubation time 2-3 minutes more. Page 12 of 16

13 B. DNA does not perform well 1. Eluate contaminated with Agarose The gel slice is not properly solubilized or is more than 400mg in weight. Vortex every 2-3 minutes during incubation time. Increase total incubation time. 2. Elution contains residual wash buffer Spin the column for 1-2 minutes extra at > 10,000 rpm to remove t h e r e s i d u a l w a s h b u f f e r completely. Page 13 of 16

14 15. Product Use Limitations mdi kits are developed and manufactured for research purpose only. The products are not recommended to be used for human, diagnostics or drug purposes for which these should be cleared by the concerned regulatory bodies in the country of use. Page 14 of 16

15 16. Product Warranty and Satisfaction Guarantee All mdi products are guaranteed and are backed by our a. Technical expertise and experience of over 30 years. b. Special mdi process for consistency and repeatability. c. Strict quality control and quality assurance regimen. d. Certificate of quality accompanied with each product. mdi provides an unconditional guarantee to replace the kit if it does not perform for any reasons other than misuse. However, the user needs to validate the performance of the kit for its specific use. Page 15 of 16

16 17. Ordering Information To order please specify as below: Type XX XX XX X Pack Size Type Code Pack Size Code MGEK MGEK Example: MGEK XX XX XX X 0250 mdi Membrane Technologies UGLMGEKKIT1505A